Crystalline form of an inhibitor of MDM2/4 and p53 interaction

ABSTRACT

A crystalline form of (S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one, which is useful in the treatment of a disease or disorder associated with the interaction between p53, or variants thereof, and MDM2 and/or MDM4, or variants thereof, respectively,

FIELD OF THE INVENTION

The present invention relates to a crystalline form of(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-onesulphate salt, which is useful in the treatment of a disease or disorderassociated with the interaction between p53, or variants thereof, andMDM2 and/or MDM4, or variants thereof, respectively, especially bindingto MDM2 and/or MDM4, or variants thereof, a process for the preparationof the crystalline form, pharmaceutical preparations comprising thecrystalline form, uses and methods of use for such crystalline form intreatment of a disease or disorder (including therapy and/orprophylaxis), and/or related subject matter as specified below.

p53 refers to all genes and/or proteins encoded thereof with the namesTP53, p53, TP73, p73, TP63, TP73L, p63. MDM2 refers to all genes and/orproteins encoded thereof with the names MDM2, Mdm2, HDM2, Hdm2. MDM4refers to all genes and/or proteins encoded thereof with the names MDM4,Mdm4, HDM4, Hdm4, MDMX, MdmX, HDMX, HdmX.

Protein p53 is known as a tumor suppressor protein which helps tocontrol cellular integrity and prevents the proliferation of permanentlydamaged cells by initiating, among other responses, growth arrest orapoptosis (controlled cell death). p53 mediates its effects in that itis a transcription factor capable of regulating a number of genes thatregulate e.g. cell cycle and apoptosis. Thus, p53 is an important cellcycle inhibitor. These activities are tightly controlled by MDM2, animportant negative regulator of the p53 tumor suppressor. “MDM2”(originally from the oncogene “murine double minute 2”) refers both tothe name of the gene as well as the protein encoded by that gene. MDM2protein functions both as an E3 ubiquitin ligase that recognizes theN-terminal trans-activation domain (TAD) of the p53 tumor suppressor andthus mediates the ubiquitin-dependent degradation of p53, and as aninhibitor of p53 transcriptional activation.

The original mouse oncogene, which codes for the MDM2 protein, wasoriginally cloned from a transformed mouse cell line. The humanhomologue of this protein was later identified and is sometimes alsocalled HDM2 (for “human double minute 2”). Further supporting the roleof MDM2 as an oncogene, several human tumor and proliferative diseasetypes have been shown to have increased levels of MDM2, including interalia soft tissue sarcomas, bone cancer, e.g. osteosarcomas, breasttumors, bladder cancer, Li-Fraumeni syndrome, brain tumor,rhabdomyosarcoma and adrenocortical carcinoma and the like. Anotherprotein belonging to the MDM2 family is MDM4, also known as MDMX.

Dysregulation of the MDM2/p53 ratio, e.g. due to mutations,polymorphisms or molecular defects in the affected cells, can thus befound in many proliferative diseases. MDM2, in view of its mentionedeffects, is capable to inhibit the activity of the tumor suppressorprotein p53, thus leading to loss of p53's tumor suppressor activity andinhibiting regulatory mechanisms that impede cells from uncontrolledproliferation. As a consequence, uncontrolled proliferation can takeplace, leading to tumors, leukemias or other proliferative diseases.

Reference herein to inhibition of the MDM2/p53 interaction includesMDM2/p53 interaction and/or MDM4/p53 interaction herein, in particularHdm2/p53 and/or Hdm4/p53 interaction.

There is a need for a crystalline form of(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one,which is useful in providing a pharmaceutical composition for use intreating diseases or disorders associated with MDM2/p53 interaction.

BRIEF SUMMARY OF THE INVENTION

It has been found that the crystalline form I of(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-onesulphate salt as described herein provides a significant improvement inprocessing properties compared to the free base amorphous form, andprovides improvements in solubility and stability.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment there is provided a crystalline form of(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-onesulphate salt. In particular, the sulphate salt is the bisulphate salt.

In another embodiment there is provided a crystalline form of(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-onebisulphate salt having a powder X ray diffraction pattern using Cu Kαradiation which includes the peaks: 18.8, 21.3 and 22.7 angle 2-Theta°with an error +/−0.2°.

In another embodiment the invention provides a crystalline form of(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-onebisulphate salt having a powder X ray diffraction pattern using Cu Kαradiation which is substantially the same as as the powder X raydiffraction pattern shown in FIG. 1, or in FIG. 2, herein

In further separate embodiments, the invention provides:

-   -   A crystalline form as defined in herein for use as        pharmaceutical.    -   A crystalline form as defined herein, for use in the treatment        of a disorder or a disease mediated by the activity of MDM2        and/or MDM4.    -   the use of a crystalline form as defined herein, for the        manufacture of a medicament for the treatment of a disorder or a        disease in a subject mediated by the activity of MDM2 and/or        MDM4.    -   A pharmaceutical composition comprising a therapeutically        effective amount of a crystalline form as defined herein, and        one or more pharmaceutically acceptable carriers.    -   A method of modulating MDM2 and/or MDM4 activity in a subject,        comprising the step of administering to a subject a        therapeutically effective amount of a crystalline form as        defined herein.    -   A method for the treatment of a disorder or a disease mediated        by the activity of MDM2 and/or MDM4 comprising the step of        administering to a subject a therapeutically effective amount of        a crystalline form as defined herein.    -   A crystalline form, use or method as described herein, wherein        the disorder or a disease is a proliferative disorder or        disease.    -   A crystalline form as defined herein, in combination with one or        more therapeutically active agents.

DEFINITIONS

As already indicated above, MDM2 (especially when mentioned as MDM2 orvariants thereof) generally refers to all genes and/or proteins encodedthereof with the names MDM2, Mdm2, HDM2, Hdm2, or a variant thereof.MDM4 (especially when mentioned as MDM4 or variants thereof) refers toall genes and/or proteins encoded thereof with the names MDM4, Mdm4,HDM4, Hdm4, MDMX, MdmX, HDMX, HdmX, or a variant thereof.

MDM2 specifically relates to MDM2 as described in EMBO J. 10, 1565-9,Fakharzadeh et al., 1991, a variant thereof refers to a variant thereofwhich still binds to p53 in the assay system described below (e.g. asplice variant, isoform, fragment, mutant or oncogene due to deletion,insertion and/or exchange of one or more, e.g. one to 430, of the aminoacids), corresponding to the full length proteins as originallydescribed, preferably at least with 0.5%, more preferably at least with5%, 10%, 20%, 30%, 40% or especially 50% or more of the affinity of MDM2to p53, and have at least 20%, more preferably at least 25%, sequenceidentity to MDM2 or to HDM2 as originally described or as mentionedbelow specifically. Where not mentioned otherwise, MDM2 generallyrelates to MDM2, Mdm2, HDM2 or Hdm2, or variants thereof, respectively,as just defined.

MDM4 specifically relates to MDM4 as described in Genomics 43, 34-42,Shvarts et al., 1997, a variant thereof refers to a variant thereofwhich still binds to p53 in the assay system described below (e.g. asplice variant, isoform, fragment, mutant or oncogene due to deletion,insertion and/or exchange of one or more, e.g. one to 430, of the aminoacids), corresponding to the full length proteins as originallydescribed, preferably at least with 0.5%, more preferably at least with5%, 10%, 20%, 30%, 40% or especially 50% or more of the affinity of MDM4to p53, and have at least 20%, more preferably at least 25%, sequenceidentity to MDM4, to MDMX, to HDM4 or to HDM2 as originally described oras mentioned below specifically. Where not mentioned otherwise, MDM4generally relates to MDM4, Mdm4, HDM4, Hdm4, MDMX, MdmX, HDMX or HdmX,or variants thereof, respectively, as just defined.

“Variants thereof” where mentioned means one or more variant(s).

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, and the like and combinations thereof, as would be known to thoseskilled in the art (see, for example, Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Exceptinsofar as any conventional carrier is incompatible with the activeingredient, its use in the therapeutic or pharmaceutical compositions iscontemplated.

By “combination”, there is meant either a fixed combination in onedosage unit form, or a kit of parts for the combined administrationwhere a compound of the formula (I) and a combination partner may beadministered independently at the same time or separately within timeintervals that especially allow that the combination partners show acooperative, e.g. synergistic effect.

The term “a therapeutically effective amount” of a compound of thepresent invention refers to an amount of the compound of the presentinvention that will elicit the biological or medical response of asubject, for example, reduction or inhibition of an enzyme or a proteinactivity, or ameliorate symptoms, alleviate conditions, slow or delaydisease progression, or prevent a disease, etc. In one non-limitingembodiment, the term “a therapeutically effective amount” refers to theamount of the compound of the present invention that, when administeredto a subject, is effective to (1) at least partially alleviating,inhibiting, preventing and/or ameliorating a condition, or a disorder ora disease (i) mediated by the dysregulation of the p53/MDM2 ratio, or(ii) associated with the dysregulation of the p53/MDM2 ratio, or (iii)characterized by the dysregulation of the MDM2/p53 ratio; or (2)reducing or inhibiting the activity of the p53/MDM2 interaction. Inanother non-limiting embodiment, the term “a therapeutically effectiveamount” refers to the amount of the compound of the present inventionthat, when administered to a cell, or a tissue, or a non-cellularbiological material, or a medium, is effective to at least partiallyreducing or inhibiting the p53/MDM2 interaction.

As used herein, the term “subject” refers to an animal. Typically theanimal is a mammal. A subject also refers to for example, primates(e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats,mice, fish, birds and the like. In certain embodiments, the subject is aprimate. In yet other embodiments, the subject is a human.

As used herein, the term “inhibit”, “inhibition” or “inhibiting” refersto the reduction or suppression of a given condition, symptom, ordisorder, or disease, or a significant decrease in the baseline activityof a biological activity or process.

As used herein, the term “treat”, “treating” or “treatment” of anydisease or disorder refers in one embodiment, to ameliorating thedisease or disorder (i.e., slowing or arresting or reducing thedevelopment of the disease or at least one of the clinical symptomsthereof). In another embodiment “treat”, “treating” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treat”, “treating” or “treatment” refers tomodulating the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both. In yet anotherembodiment, “treat”, “treating” or “treatment” refers to preventing ordelaying the onset or development or progression of the disease ordisorder.

As used herein, a subject is “in need of” a treatment if such subjectwould benefit biologically, medically or in quality of life from suchtreatment.

As used herein, the term “a,” “an,” “the” and similar terms used in thecontext of the present invention (especially in the context of theclaims) are to be construed to cover both the singular and plural unlessotherwise indicated herein or clearly contradicted by the context.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed.

“Sulphate salt” can mean the bisulphate salt, or the monosulphate salt,or mixtures thereof.

Pharmaceutical Compositions

The invention also relates to pharmaceutical compositions comprising acrystalline form of the present invention. The invention thus provides

-   -   a pharmaceutical composition comprising (i.e. containing or        consisting of) the crystalline form as defined herein and one or        more carriers/excipients;    -   a pharmaceutical composition comprising a therapeutically        effective amount of the crystalline form as defined herein, and        one or more pharmaceutically acceptable carriers/excipients.

The present invention provides a pharmaceutical composition comprising athe crystalline form of the present invention and a pharmaceuticallyacceptable carrier. The pharmaceutical composition can be formulated forparticular routes of administration such as oral administration,parenteral administration, and rectal administration, etc. In addition,the pharmaceutical compositions of the present invention can be made upin a solid form (including without limitation capsules, tablets, pills,granules, powders or suppositories), or in a liquid form (includingwithout limitation solutions, suspensions or emulsions). Thepharmaceutical compositions can be subjected to conventionalpharmaceutical operations such as sterilization and/or can containconventional inert diluents, lubricating agents, or buffering agents, aswell as adjuvants, such as preservatives, stabilizers, wetting agents,emulsifiers and buffers, etc.

Typically, the pharmaceutical compositions are tablets or gelatincapsules comprising the active ingredient together with

a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol,cellulose and/or glycine;b) lubricants, e.g., silica, talcum, stearic acid, its magnesium orcalcium salt and/or polyethyleneglycol; for tablets alsoc) binders, e.g., magnesium aluminum silicate, starch paste, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone; if desiredd) disintegrants, e.g., starches, agar, alginic acid or its sodium salt,or effervescent mixtures; and/ore) absorbents, colorants, flavors and sweeteners.

Tablets may be either film coated or enteric coated according to methodsknown in the art.

Suitable compositions for oral administration include an effectiveamount of a compound of the invention in the form of tablets, lozenges,aqueous or oily suspensions, dispersible powders or granules, emulsion,hard or soft capsules, or syrups or elixirs. Compositions intended fororal use are prepared according to any method known in the art for themanufacture of pharmaceutical compositions and such compositions cancontain one or more agents selected from the group consisting ofsweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets may contain the active ingredient in admixturewith nontoxic pharmaceutically acceptable excipients which are suitablefor the manufacture of tablets. These excipients are, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for example,starch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets are uncoated or coated byknown techniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate can be employed. Formulations fororal use can be presented as hard gelatin capsules wherein the activeingredient is mixed with an inert solid diluent, for example, calciumcarbonate, calcium phosphate or kaolin, or as soft gelatin capsuleswherein the active ingredient is mixed with water or an oil medium, forexample, peanut oil, liquid paraffin or olive oil.

Certain injectable compositions are aqueous isotonic solutions orsuspensions, and suppositories are advantageously prepared from fattyemulsions or suspensions. Said compositions may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, they may also contain othertherapeutically valuable substances. Said compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and contain about 0.1-75%, or contain about 1-50%, of theactive ingredient.

Suitable compositions for transdermal application include an effectiveamount of a compound of the invention with a suitable carrier. Carrierssuitable for transdermal delivery include absorbable pharmacologicallyacceptable solvents to assist passage through the skin of the host. Forexample, transdermal devices are in the form of a bandage comprising abacking member, a reservoir containing the compound optionally withcarriers, optionally a rate controlling barrier to deliver the compoundof the skin of the host at a controlled and predetermined rate over aprolonged period of time, and means to secure the device to the skin.

Suitable compositions for topical application, e.g., to the skin andeyes, include aqueous solutions, suspensions, ointments, creams, gels orsprayable formulations, e.g., for delivery by aerosol or the like. Suchtopical delivery systems will in particular be appropriate for dermalapplication, e.g., for the treatment of skin cancer, e.g., forprophylactic use in sun creams, lotions, sprays and the like. They arethus particularly suited for use in topical, including cosmetic,formulations well-known in the art. Such may contain solubilizers,stabilizers, tonicity enhancing agents, buffers and preservatives.

As used herein a topical application may also pertain to an inhalationor to an intranasal application. They may be conveniently delivered inthe form of a dry powder (either alone, as a mixture, for example a dryblend with lactose, or a mixed component particle, for example withphospholipids) from a dry powder inhaler or an aerosol spraypresentation from a pressurised container, pump, spray, atomizer ornebuliser, with or without the use of a suitable propellant.

The present invention further provides anhydrous pharmaceuticalcompositions and dosage forms comprising the compounds of the presentinvention as active ingredients, since water may facilitate thedegradation of certain compounds.

Anhydrous pharmaceutical compositions and dosage forms of the inventioncan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. An anhydrous pharmaceuticalcomposition may be prepared and stored such that its anhydrous nature ismaintained. Accordingly, anhydrous compositions are packaged usingmaterials known to prevent exposure to water such that they can beincluded in suitable formulary kits. Examples of suitable packaginginclude, but are not limited to, hermetically sealed foils, plastics,unit dose containers (e.g., vials), blister packs, and strip packs.

The invention further provides pharmaceutical compositions and dosageforms that comprise one or more agents that reduce the rate by which thecompound of the present invention as an active ingredient willdecompose. Such agents, which are referred to herein as “stabilizers,”include, but are not limited to, antioxidants such as ascorbic acid, pHbuffers, or salt buffers, etc.

Pharmaceutical Uses

The invention relates in another aspect to the use of the crystallineform of the present invention as pharmaceuticals. The invention thusprovides:

-   -   the crystalline form as defined herein for use as a medicament;    -   the crystalline form of the present invention for use in the        treatment of a disorder or a disease in a subject associated        with the activity of MDM2 and/or MDM4;    -   the use of the crystalline form of the present invention, in the        manufacture of a medicament for the treatment of a disorder or a        disease in a subject associated with the activity of MDM2 and/or        MDM4;    -   the use of the crystalline form as defined herein, for the        treatment of a proliferative disorder or disease selected from        cancer or tumor diseases, such as benign or malignant tumors, a        sarcoma, such as liposarcoma, rhabdomyosarcoma or bone cancer,        e.g. osteosarcomas, a carcinoma, such as of the brain, kidney,        liver, adrenal gland, bladder, breast, gastric, ovary, colon,        rectum, prostate, pancreas, lung, vagina or thyroid, a        glioblastoma, a multiple myeloma, a gastrointestinal cancer,        especially colon carcinoma or colorectal adenoma, a tumor of the        head and neck, a melanoma, a prostate hyperplasia, a neoplasia,        a neoplasia of epithelial character, a leukemia or a lymphoma,        such as of B- or T-cell origin, and metastases in other organs),        viral infections (e.g. herpes, papilloma, HIV, Kaposi's, viral        hepatitis);    -   a method of modulating of MDM2 and/or MDM4 activity in a        subject, comprising the step of administering to a subject a        therapeutically effective amount of the crystalline form of the        present invention;    -   a method for the treatment of a disorder or a disease associated        with the activity of MDM2 and/or MDM4 comprising the step of        administering to a subject a therapeutically effective amount of        the crystalline form of the present invention as described        herein;

The efficacy of(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-onecan be demonstrated as shown in WO 98/01467 (which especially regardingthe assays is included herein by reference) or preferably follows:

Time Resolved Fluorescence Energy Transfer (TR-FRET) Assay

The inhibition of p53-Hdm2 and p53-Hdm4 interactions is measured by timeresolved fluorescence energy transfer (TR-FRET). Fluorescence energytransfer (or Foerster resonance energy transfer) describes an energytransfer between donor and acceptor fluorescent molecules. For thisassay, MDM2 protein (amino acids 2-188) and MDM4 protein (amino acids2-185), tagged with a C-terminal Biotin moiety, are used in combinationwith a Europium labeled streptavidin (Perkin Elmer, Inc., Waltham,Mass., USA) serving as the donor fluorophore. The p53 derived, Cy5labeled peptide Cy5-TFSDLWKLL (p53 aa18-26) is the energy acceptor. Uponexcitation of the donor molecule at 340 nm, binding interaction betweenMDM2 or MDM4 and the p53 peptide induces energy transfer and enhancedresponse at the acceptor emission wavelength at 665 nm. Disruption ofthe formation of the p53-MDM2 or p53-MDM4 complex due to an inhibitormolecule binding to the p53 binding site of MDM2 or MDM4 results inincreased donor emission at 615 nm. The ratiometric FRET assay readoutis calculated from the raw data of the two distinct fluorescence signalsmeasured in time resolved mode (countrate 665 nm/countrate 615 nm×1000).

The test is performed in white 1536 w microtiterplates (Greiner Bio-OneGmbH, Frickenhausen, Germany) in a total volume of 3.1 μl by combining100 nl of compounds diluted in 90% DMSO/10% H₂O (3.2% final DMSOconcentration) with 2 μl Europium labeled streptavidin (finalconcentration 2.5 nM) in reaction buffer (PBS, 125 mM NaCl, 0.001%Novexin (consists of carbohydrate polymers (Novexin polymers), designedto increase the solubility and stability of proteins; Novexin Ltd.,Cambridgeshire, United Kingdom), Gelatin 0.01%, 0.2% Pluronic (blockcopolymer from ethylenoxide and propyleneoxide, BASF, Ludwigshafen,Germany), 1 mM DTT), followed by the addition of 0.5 μl MDM2-Bio orMDM4-Bio diluted in assay buffer (final concentration 10 nM). Allow thesolution to pre-incubate for 15 minutes at room temperature, followed byaddition of 0.5 μl Cy5-p53 peptide in assay buffer (final concentration20 nM). Incubate at room temperature for 10 minutes prior to reading theplate. For measurement of samples, an Analyst GT multimode microplatereader (Molecular Devices) with the following settings is used: Dichroicmirror 380 nm, Excitation 330 nm, Emission Donor 615 nm and EmissionAcceptor 665 nm. 1050 values are calculated by curve fitting usingXLfit. If not specified, reagents are purchased from Sigma Chemical Co,St. Louis, Mo., USA.

There are also experiments that can demonstrate the antitumor activityof compounds of the formula (I) in vivo.

For example, female Harlan (Indianapolis, Ind., USA) athymic nu/nu micewith s.c. transplanted human osteosarcoma SJSA-1 tumors can be used todetermine the anti-tumor activity of p53/MDM2 interaction inhibitors. Onday 0, with the animals under peroral Forene®(1-chloro-2,2,2-trifluoroethyldifluormethylether, Abbot, Wiesbaden,Germany) narcosis, 3×10⁶ cells are injected under the skin on theanimals' left flank. When tumors reach a volume of 100 mm³, the mice aredivided at random into groups of 6-8 animals and treatment commences.The treatment is carried out for a 2-3 weeks period with peroral,intravenous or intra-peritoneal administration twice daily (or lessfrequently) of a compound of the formula (I) in a suitable vehicle atdefined doses. The tumors are measured twice a week with a slide gaugeand the volume of the tumors is calculated.

As an alternative to cell line SJSA-1, other cell lines may also be usedin the same manner, for example,

-   -   the HCT116 colon carcinoma cell line (ATCC No. CCL-247);    -   the LNCaP clone FGC prostate carcinoma cell line (ATCC No.        CRL-1740);    -   the RKO colon carcinoma cell line (ATCC No. CRL-2577);    -   the HT1080 fibrosarcoma cell line (ATCC No. CCL-121);    -   the A375 malignant melanoma cell line (ATCC No. CRL-1619),    -   the NCI-H460 large cell lung carcinoma cell line (ATCC No.        HTB-177);    -   the JEG-3 choriocarcinoma (ATCC No. HTB-36)    -   the ZR-75-1 breast ductal carcinoma (ATCC No. CRL-1500)

((S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-onefree base:

IC₅₀ (μM) of p53-Hdm2 inhibition (TR-FRET) Assay: 0.0008IC₅₀ (μM) of p53-Hdm4 inhibition (TR-FRET) Assay: 2.10

((S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-onebisulphate salt as disclosed herein:

IC₅₀ (μM) of p53-Hdm2 inhibition (TR-FRET) Assay: 0.0019IC₅₀ (μM) of p53-Hdm4 inhibition (TR-FRET) Assay: 2.2

Combinations

The invention relates in another aspect to combinations comprising thecrystalline form of(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-oneas described herein, and one or more additional active ingredients. Theinvention thus provides:

-   -   a combination in particular a pharmaceutical combination,        comprising the crystalline form of        (S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one        as described herein, and one or more therapeutically active        agents, particularly antiproliferative agents;    -   a combined pharmaceutical composition, adapted for simultaneous        or sequential administration, comprising the crystalline form of        (S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one        as described herein; therapeutically effective amount(s) of one        or more combination partners, particularly antiproliferative        agents; one or more pharmaceutically acceptable excepients;    -   a combined pharmaceutical composition as defined herein (i) as        pharmaceutical, (ii) for use in the treatment of a disorder or a        disease mediated by the activity of MDM2 and/or MDM4, (iii) in a        method of treatment of a disorder or a disease mediated by the        activity of MDM2 and/or MDM4.

The crystalline form of(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-oneas described herein may also be used to advantage in combination withother antiproliferative compounds. Such antiproliferative compoundsinclude, but are not limited to aromatase inhibitors; antiestrogens;topoisomerase I inhibitors; topoisomerase II inhibitors; microtubuleactive compounds; alkylating compounds; histone deacetylase inhibitors;compounds which induce cell differentiation processes; cyclooxygenaseinhibitors; MMP inhibittors; mTOR inhibitors, such as RAD001;antineoplastic antimetabolites; platin compounds; compoundstargeting/decreasing a protein or lipid kinase activity and furtheranti-angiogenic compounds; compounds which target, decrease or inhibitthe activity of a protein or lipid phosphatase; gonadorelin agonists;anti-androgens; methionine aminopeptidase inhibitors; bisphosphonates;biological response modifiers; antiproliferative antibodies, such asHCD122; heparanase inhibitors; inhibitors of Ras oncogenic isoforms;telomerase inhibitors; proteasome inhibitors; compounds used in thetreatment of hematologic malignancies, such as fludarabine; compoundswhich target, decrease or inhibit the activity of Flt-3, such as PKC412;Hsp90 inhibitors such as 17-AAG (17-allylaminogeldanamycin, NSC330507),17-DMAG (17-dimethylaminoethylamino-17-demethoxy-geldanamycin,NSC707545), IPI-504, CNF1010, CNF2024, CNF1010 from ConformaTherapeutics and AUY922; temozolomide (TEMODAL™); kinesin spindleprotein inhibitors, such as SB715992 or SB743921 from GlaxoSmithKline,or pentamidine/chlorpromazine from CombinatoRx; PI3K inhibitors, such asBEZ235; RAF inhibitors, such as RAF265; MEK inhibitors such as ARRY142886 from Array PioPharma, AZD6244 from AstraZeneca, PD181461 fromPfizer, leucovorin, EDG binders, antileukemia compounds, ribonucleotidereductase inhibittors, S-adenosylmethionine decarboxylase inhibitors,regulators of apoptosis, antiproliferative antibodies or otherchemotherapeutic compounds. Further, alternatively or in addition theymay be used in combination with other tumor treatment approaches,including surgery, ionizing radiation, photodynamic therapy, implants,e.g. with corticosteroids, hormones, or they may be used asradiosensitizers. Also, in anti-inflammatory and/or antiproliferativetreatment, combination with anti-inflammatory drugs is included.Combination is also possible with antihistamine drug substances,bronchodilatatory drugs, NSAID or antagonists of chemokine receptors.

The term “aromatase inhibitor” as used herein relates to a compoundwhich inhibits the estrogen production, i.e. the conversion of thesubstrates androstenedione and testosterone to estrone and estradiol,respectively. The term includes, but is not limited to steroids,especially atamestane, exemestane and formestane and, in particular,non-steroids, especially aminoglutethimide, roglethimide,pyridoglutethimide, trilostane, testolactone, ketokonazole, vorozole,fadrozole, anastrozole and letrozole. Exemestane can be administered,e.g., in the form as it is marketed, e.g. under the trademark AROMASIN.Formestane can be administered, e.g., in the form as it is marketed,e.g. under the trademark LENTARON. Fadrozole can be administered, e.g.,in the form as it is marketed, e.g. under the trademark AFEMA.Anastrozole can be administered, e.g., in the form as it is marketed,e.g. under the trademark ARIMIDEX. Letrozole can be administered, e.g.,in the form as it is marketed, e.g. under the trademark FEMARA or FEMAR.Aminoglutethimide can be administered, e.g., in the form as it ismarketed, e.g. under the trademark ORIMETEN. A combination of theinvention comprising a chemotherapeutic agent which is an aromataseinhibitor is particularly useful for the treatment of hormone receptorpositive tumors, e.g. breast tumors.

The term “antiestrogen” as used herein relates to a compound whichantagonizes the effect of estrogens at the estrogen receptor level. Theterm includes, but is not limited to tamoxifen, fulvestrant, raloxifeneand raloxifene hydrochloride. Tamoxifen can be administered, e.g., inthe form as it is marketed, e.g. under the trademark NOLVADEX.Raloxifene hydrochloride can be administered, e.g., in the form as it ismarketed, e.g. under the trademark EVISTA. Fulvestrant can be formulatedas disclosed in U.S. Pat. No. 4,659,516 or it can be administered, e.g.,in the form as it is marketed, e.g. under the trademark FASLODEX. Acombination of the invention comprising a chemotherapeutic agent whichis an antiestrogen is particularly useful for the treatment of estrogenreceptor positive tumors, e.g. breast tumors.

The term “anti-androgen” as used herein relates to any substance whichis capable of inhibiting the biological effects of androgenic hormonesand includes, but is not limited to, bicalutamide (CASODEX™), which canbe formulated, e.g. as disclosed in U.S. Pat. No. 4,636,505.

The term “gonadorelin agonist” as used herein includes, but is notlimited to abarelix, goserelin and goserelin acetate. Goserelin isdisclosed in U.S. Pat. No. 4,100,274 and can be administered, e.g., inthe form as it is marketed, e.g. under the trademark ZOLADEX. Abarelixcan be formulated, e.g. as disclosed in U.S. Pat. No. 5,843,901.

The term “topoisomerase I inhibitor” as used herein includes, but is notlimited to topotecan, gimatecan, irinotecan, camptothecian and itsanalogues, 9-nitrocamptothecin and the macromolecular camptothecinconjugate PNU-166148 (compound A1 in WO99/17804). Irinotecan can beadministered, e.g. in the form as it is marketed, e.g. under thetrademark CAMPTOSAR. Topotecan can be administered, e.g., in the form asit is marketed, e.g. under the trademark HYCAMTIN.

The term “topoisomerase II inhibitor” as used herein includes, but isnot limited to the anthracyclines such as doxorubicin (includingliposomal formulation, e.g. CAELYX), daunorubicin, epirubicin,idarubicin and nemorubicin, the anthraquinones mitoxantrone andlosoxantrone, and the podophillotoxines etoposide and teniposide.Etoposide can be administered, e.g. in the form as it is marketed, e.g.under the trademark ETOPOPHOS. Teniposide can be administered, e.g. inthe form as it is marketed, e.g. under the trademark VM 26-BRISTOL.Doxorubicin can be administered, e.g. in the form as it is marketed,e.g. under the trademark ADRIBLASTIN or ADRIAMYCIN. Epirubicin can beadministered, e.g. in the form as it is marketed, e.g. under thetrademark FARMORUBICIN. Idarubicin can be administered, e.g. in the formas it is marketed, e.g. under the trademark ZAVEDOS. Mitoxantrone can beadministered, e.g. in the form as it is marketed, e.g. under thetrademark NOVANTRON.

The term “microtubule active compound” relates to microtubulestabilizing, microtubule destabilizing compounds and microtublinpolymerization inhibitors including, but not limited to taxanes, e.g.paclitaxel and docetaxel, vinca alkaloids, e.g., vinblastine, especiallyvinblastine sulfate, vincristine especially vincristine sulfate, andvinorelbine, discodermolides, cochicine and epothilones and derivativesthereof, e.g. epothilone B or D or derivatives thereof. Paclitaxel maybe administered e.g. in the form as it is marketed, e.g. TAXOL™.Docetaxel can be administered, e.g., in the form as it is marketed, e.g.under the trademark TAXOTERE. Vinblastine sulfate can be administered,e.g., in the form as it is marketed, e.g. under the trademark VINBLASTINR.P. Vincristine sulfate can be administered, e.g., in the form as it ismarketed, e.g. under the trademark FARMISTIN. Discodermolide can beobtained, e.g., as disclosed in U.S. Pat. No. 5,010,099. Also includedare Epothilone derivatives which are disclosed in WO 98/10121, U.S. Pat.No. 6,194,181, WO 98/25929, WO 98/08849, WO 99/43653, WO 98/22461 and WO00/31247. Especially preferred are Epothilone A and/or B.

The term “alkylating compound” as used herein includes, but is notlimited to, cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNUor Gliadel). Cyclophosphamide can be administered, e.g., in the form asit is marketed, e.g. under the trademark CYCLOSTIN. Ifosfamide can beadministered, e.g., in the form as it is marketed, e.g. under thetrademark HOLOXAN.

The term “antineoplastic antimetabolite” includes, but is not limitedto, 5-Fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylatingcompounds, such as 5-azacytidine and decitabine, methotrexate andedatrexate, and folic acid antagonists such as pemetrexed. Capecitabinecan be administered, e.g., in the form as it is marketed, e.g. under thetrademark XELODA. Gemcitabine can be administered, e.g., in the form asit is marketed, e.g. under the trademark GEMZAR.

The term “platin compound” as used herein includes, but is not limitedto, carboplatin, cis-platin, cisplatinum and oxaliplatin. Carboplatincan be administered, e.g., in the form as it is marketed, e.g. under thetrademark CARBOPLAT. Oxaliplatin can be administered, e.g., in the formas it is marketed, e.g. under the trademark ELOXATIN.

The term “compounds targeting/decreasing a protein or lipid kinaseactivity”; or a “protein or lipid phosphatase activity”; or “furtheranti-angiogenic compounds” as used herein includes, but is not limitedto, protein tyrosine kinase and/or serine and/or threonine kinaseinhibitors or lipid kinase inhibitors, e.g.,

a) compounds targeting, decreasing or inhibiting the activity of theplatelet-derived growth factor-receptors (PDGFR), such as compoundswhich target, decrease or inhibit the activity of PDGFR, especiallycompounds which inhibit the PDGF receptor, e.g. aN-phenyl-2-pyrimidine-amine derivative, e.g. imatinib, SU101, SU6668 andGFB-111;b) compounds targeting, decreasing or inhibiting the activity of thefibroblast growth factor-receptors (FGFR);c) compounds targeting, decreasing or inhibiting the activity of theinsulin-like growth factor receptor I (IGF-IR), such as compounds whichtarget, decrease or inhibit the activity of IGF-IR, especially compoundswhich inhibit the kinase activity of IGF-I receptor, such as thosecompounds disclosed in WO 02/092599, or antibodies that target theextracellular domain of IGF-I receptor or its growth factors;d) compounds targeting, decreasing or inhibiting the activity of the Trkreceptor tyrosine kinase family, or ephrin B4 inhibitors;e) compounds targeting, decreasing or inhibiting the activity of the Axlreceptor tyrosine kinase family;f) compounds targeting, decreasing or inhibiting the activity of the Retreceptor tyrosine kinase;g) compounds targeting, decreasing or inhibiting the activity of theKit/SCFR receptor tyrosine kinase, i.e C-kit receptor tyrosinekinases—(part of the PDGFR family), such as compounds which target,decrease or inhibit the activity of the c-Kit receptor tyrosine kinasefamily, especially compounds which inhibit the c-Kit receptor, e.g.imatinib;h) compounds targeting, decreasing or inhibiting the activity of membersof the c-Abl family, their gene-fusion products (e.g. BCR-Abl kinase)and mutants, such as compounds which target decrease or inhibit theactivity of c-Abl family members and their gene fusion products, e.g. aN-phenyl-2-pyrimidine-amine derivative, e.g. imatinib or nilotinib(AMN107); PD180970; AG957; NSC 680410; PD173955 from ParkeDavis; ordasatinib (BMS-354825)i) compounds targeting, decreasing or inhibiting the activity of membersof the protein kinase C (PKC) and Raf family of serine/threoninekinases, members of the MEK, SRC, JAK, FAK, PDK1, PKB/Akt, and Ras/MAPKfamily members, and/or members of the cyclin-dependent kinase family(CDK) and are especially those staurosporine derivatives disclosed inU.S. Pat. No. 5,093,330, e.g. midostaurin; examples of further compoundsinclude e.g. UCN-01, safingol, BAY 43-9006, Bryostatin 1, Perifosine;Ilmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521;LY333531/LY379196; isochinoline compounds such as those disclosed in WO00/09495; FTIs; BEZ235 (a PI3K inhibitor) or AT7519 (CDK inhibitor);j) compounds targeting, decreasing or inhibiting the activity ofprotein-tyrosine kinase inhibitors, such as compounds which target,decrease or inhibit the activity of protein-tyrosine kinase inhibitorsinclude imatinib mesylate (GLEEVEC™) or tyrphostin. A tyrphostin ispreferably a low molecular weight (Mr<1500) compound, or apharmaceutically acceptable salt thereof, especially a compound selectedfrom the benzylidenemalonitrile class or the S-arylbenzenemalonirile orbisubstrate quinoline class of compounds, more especially any compoundselected from the group consisting of Tyrphostin A23/RG-50810; AG 99;Tyrphostin AG 213; Tyrphostin AG 1748; Tyrphostin AG 490; TyrphostinB44; Tyrphostin B44 (+) enantiomer; Tyrphostin AG 555; AG 494;Tyrphostin AG 556, AG957 and adaphostin(4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester;NSC 680410, adaphostin);k) compounds targeting, decreasing or inhibiting the activity of theepidermal growth factor family of receptor tyrosine kinases (EGFR,ErbB2, ErbB3, ErbB4 as homo- or heterodimers) and their mutants, such ascompounds which target, decrease or inhibit the activity of theepidermal growth factor receptor family are especially compounds,proteins or antibodies which inhibit members of the EGF receptortyrosine kinase family, e.g. EGF receptor, ErbB2, ErbB3 and ErbB4 orbind to EGF or EGF related ligands, and are in particular thosecompounds, proteins or monoclonal antibodies generically andspecifically disclosed in WO 97/02266, e.g. the compound of ex. 39, orin EP 0 564 409, WO 99/03854, EP 0520722, EP 0 566 226, EP 0 787 722, EP0 837 063, U.S. Pat. No. 5,747,498, WO 98/10767, WO 97/30034, WO97/49688, WO 97/38983 and, especially, WO 96/30347 (e.g. compound knownas CP 358774), WO 96/33980 (e.g. compound ZD 1839) and WO 95/03283 (e.g.compound ZM105180); e.g. trastuzumab (Herceptin™) cetuximab (Erbitux™),Iressa, Tarceva, OSI-774, CI-1033, EKB-569, GW-2016, E1.1, E2.4, E2.5,E6.2, E6.4, E2.11, E6.3 or E7.6.3, and 7H-pyrrolo-[2,3-d]pyrimidinederivatives which are disclosed in WO 03/013541; andl) compounds targeting, decreasing or inhibiting the activity of thec-Met receptor, such as compounds which target, decrease or inhibit theactivity of c-Met, especially compounds which inhibit the kinaseactivity of c-Met receptor, or antibodies that target the extracellulardomain of c-Met or bind to HGF;m) compounds targeting, decreasing or inhibiting the activity of PI3K,such as BEZ235 or BKM120;n) compounds targeting, decreasing or inhibiting the activity of thecyclin dependent kinase family, such as PD 0332991.

Further anti-angiogenic compounds include compounds having anothermechanism for their activity, e.g. unrelated to protein or lipid kinaseinhibition e.g. thalidomide (THALOMID) and TNP-470.

Compounds which target, decrease or inhibit the activity of a protein orlipid phosphatase are e.g. inhibitors of phosphatase 1, phosphatase 2A,or CDC25, e.g. okadaic acid or a derivative thereof.

Compounds which induce cell differentiation processes are e.g. retinoicacid, α- γ- or δ-tocopherol or α- γ- or δ-tocotrienol.

The term cyclooxygenase inhibitor as used herein includes, but is notlimited to, e.g. Cox-2 inhibitors, 5-alkyl substituted2-arylaminophenylacetic acid and derivatives, such as celecoxib(CELEBREX™), rofecoxib (VIOXX™), etoricoxib, valdecoxib or a5-alkyl-2-arylaminophenylacetic acid, e.g.5-methyl-2-(2′-chloro-6′-fluoroanilino)phenyl acetic acid, lumiracoxib.

The term “bisphosphonates” as used herein includes, but is not limitedto, etridonic, clodronic, tiludronic, pamidronic, alendronic,ibandronic, risedronic and zoledronic acid. “Etridonic acid” can beadministered, e.g., in the form as it is marketed, e.g. under thetrademark DIDRONEL. “Clodronic acid” can be administered, e.g., in theform as it is marketed, e.g. under the trademark BONEFOS. “Tiludronicacid” can be administered, e.g., in the form as it is marketed, e.g.under the trademark SKELID. “Pamidronic acid” can be administered, e.g.in the form as it is marketed, e.g. under the trademark AREDIA.“Alendronic acid” can be administered, e.g., in the form as it ismarketed, e.g. under the trademark FOSAMAX. “Ibandronic acid” can beadministered, e.g., in the form as it is marketed, e.g. under thetrademark BONDRANAT. “Risedronic acid” can be administered, e.g., in theform as it is marketed, e.g. under the trademark ACTONEL. “Zoledronicacid” can be administered, e.g. in the form as it is marketed, e.g.under the trademark ZOMETA.

The term “mTOR inhibitors” relates to compounds which inhibit themammalian target of rapamycin (mTOR) and which possess antiproliferativeactivity such as sirolimus (Rapamune™), everolimus (Certican™ orAfinitor™), CCI-779 and ABT578.

The term “heparanase inhibitor” as used herein refers to compounds whichtarget, decrease or inhibit heparin sulfate degradation. The termincludes, but is not limited to, PI-88.

The term “biological response modifier” as used herein refers to alymphokine or interferons, e.g. interferon γ.

The term “inhibitor of Ras oncogenic isoforms”, e.g. H-Ras, K-Ras, orN-Ras, as used herein refers to compounds which target, decrease orinhibit the oncogenic activity of Ras e.g. a “farnesyl transferaseinhibitor” e.g. L-744832, DK8G557 or R115777 (Zarnestra).

The term “telomerase inhibitor” as used herein refers to compounds whichtarget, decrease or inhibit the activity of telomerase. Compounds whichtarget, decrease or inhibit the activity of telomerase are especiallycompounds which inhibit the telomerase receptor, e.g. telomestatin.

The term “methionine aminopeptidase inhibitor” as used herein refers tocompounds which target, decrease or inhibit the activity of methionineaminopeptidase. Compounds which target, decrease or inhibit the activityof methionine aminopeptidase are e.g. bengamide or a derivative thereof.

The term “proteasome inhibitor” as used herein refers to compounds whichtarget, decrease or inhibit the activity of the proteasome. Compoundswhich target, decrease or inhibit the activity of the proteasome includee.g. Bortezomid (Velcade™) and MLN 341.

The term “matrix metalloproteinase inhibitor” or (“MMP” inhibitor) asused herein includes, but is not limited to, collagen peptidomimetic andnonpeptidomimetic inhibitors, tetrazolyle derivatives, e.g. hydroxamatepeptidomimetic inhibitor batimastat and its orally bioavailable analoguemarimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551)BMS-279251, BAY 12-9566, TAA211, MMI270B or AAJ996.

The term “compounds used in the treatment of hematologic malignancies”as used herein includes, but is not limited to, FMS-like tyrosine kinaseinhibitors e.g. compounds targeting, decreasing or inhibiting theactivity of FMS-like tyrosine kinase receptors (Flt-3R); interferon,1-b-D-arabinofuransylcytosine (ara-c) and bisulfan; and ALK inhibitorse.g. compounds which target, decrease or inhibit anaplastic lymphomakinase.

Compounds which target, decrease or inhibit the activity of FMS-liketyrosine kinase receptors (Flt-3R) are especially compounds, proteins orantibodies which inhibit members of the Flt-3R receptor kinase family,e.g. PKC412, TKI258, midostaurin, a staurosporine derivative, SU11248and MLN518.

The term “HSP90 inhibitors” as used herein includes, but is not limitedto, compounds targeting, decreasing or inhibiting the intrinsic ATPaseactivity of HSP90; degrading, targeting, decreasing or inhibiting theHSP90 client proteins via the ubiquitin proteosome pathway. Compoundstargeting, decreasing or inhibiting the intrinsic ATPase activity ofHSP90 are especially compounds, proteins or antibodies which inhibit theATPase activity of HSP90 e.g., 17-allylamino,17-demethoxygeldanamycin(17AAG), a geldanamycin derivative; other geldanamycin relatedcompounds; radicicol and HDAC inhibitors. An example HSP90 inhibitor isAUY922.

The term “regulators of apoptosis” as used herein includes, but is notlimited to, compounds targeting, decreasing or inhibiting the activityof Bcl2 family members (such as ABT-263) and IAP family members (such asAEG40826); or inducing apoptosis by known or unknown mechanism(s) ofaction (e.g. TRAIL antibody, DR5 antibody).

The term “antiproliferative antibodies” as used herein includes, but isnot limited to, trastuzumab (Herceptin™), Trastuzumab-DM1, erbitux,bevacizumab (Avastin™) rituximab (Rituxan™), PRO64553 (anti-CD40), 2C4Antibody and HCD122 antibody (anti-CD40). By antibodies is meant e.g.intact monoclonal antibodies, polyclonal antibodies, multispecificantibodies formed from at least 2 intact antibodies, and antibodiesfragments so long as they exhibit the desired biological activity.

For the treatment of acute myeloid leukemia (AML), the solid form of theinvention herein can be used in combination with standard leukemiatherapies, especially in combination with therapies used for thetreatment of AML. In particular, said solid form can be administered incombination with, e.g., farnesyl transferase inhibitors and/or otherdrugs useful for the treatment of AML, such as Daunorubicin, Adriamycin,Ara-C, VP-16, Teniposide, Mitoxantrone, Idarubicin, Carboplatinum andPKC412.

The term “antileukemic compounds” includes, for example, Ara-C, apyrimidine analog, which is the 2″-alpha-hydroxy ribose (arabinoside)derivative of deoxycytidine. Also included is the purine analog ofhypoxanthine, 6-mercaptopurine (6-MP) and fludarabine phosphate.

Compounds which target, decrease or inhibit activity of histonedeacetylase (HDAC) inhibitors such as sodium butyrate andsuberoylanilide hydroxamic acid (SAHA) inhibit the activity of theenzymes known as histone deacetylases. Specific HDAC inhibitors includeMS275, SAHA, FK228 (formerly FR901228), Trichostatin A, LDH589 disclosedin WO 02/22577 and compounds disclosed in U.S. Pat. No. 6,552,065, inparticular,N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide,or a pharmaceutically acceptable salt thereof andN-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl}-2E-2-propenamide,or a pharmaceutically acceptable salt thereof, especially the lactatesalt.

Somatostatin receptor antagonists as used herein refer to compoundswhich target, treat or inhibit the somatostatin receptor such asoctreotide, and SOM230 (pasireotide).

Tumor cell damaging approaches refer to approaches such as ionizingradiation. The term “ionizing radiation” referred to above andhereinafter means ionizing radiation that occurs as eitherelectromagnetic rays (such as X-rays and gamma rays) or particles (suchas alpha and beta particles). Ionizing radiation is provided in, but notlimited to, radiation therapy and is known in the art. See Hellman,Principles of Radiation Therapy, Cancer, in Principles and Practice ofOncology, Devita et al., Eds., 4^(th) Edition, Vol. 1, pp. 248-275(1993).

The term “EDG binders” as used herein refers a class ofimmunosuppressants that modulates lymphocyte recirculation, such asFTY720.

The term “ribonucleotide reductase inhibitors” refers to pyrimidine orpurine nucleoside analogs including, but not limited to, fludarabineand/or cytosine arabinoside (ara-C), 6-thioguanine, 5-fluorouracil,cladribine, 6-mercaptopurine (especially in combination with ara-Cagainst ALL) and/or pentostatin. Ribonucleotide reductase inhibitors areespecially hydroxyurea or 2-hydroxy-1H-isoindole-1,3-dione derivatives,such as PL-1, PL-2, PL-3, PL-4, PL-5, PL-6, PL-7 or PL-8 mentioned inNandy et al., Acta Oncologica, Vol. 33, No. 8, pp. 953-961 (1994).

The term “S-adenosylmethionine decarboxylase inhibitors” as used hereinincludes, but is not limited to the compounds disclosed in U.S. Pat. No.5,461,076.

Also included are in particular those compounds, proteins or monoclonalantibodies of VEGF disclosed in WO 98/35958, e.g.1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharmaceuticallyacceptable salt thereof, e.g. the succinate, or in WO 00/09495, WO00/27820, WO 00/59509, WO 98/11223, WO 00/27819 and EP 0 769 947; thoseas described by Prewett et al, Cancer Res, Vol. 59, pp. 5209-5218(1999); Yuan et al., Proc Natl Aced Sci USA, Vol. 93, pp. 14765-14770(1996); Zhu et al., Cancer Res, Vol. 58, pp. 3209-3214 (1998); andMordenti et al., Toxicol Pathol, Vol. 27, No. 1, pp. 14-21 (1999); in WO00/37502 and WO 94/10202; ANGIOSTATIN, described by O'Reilly et al.,Cell, Vol. 79, pp. 315-328 (1994); ENDOSTATIN, described by O'Reilly etal., Cell, Vol. 88, pp. 277-285 (1997); anthranilic acid amides; ZD4190;ZD6474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies oranti-VEGF receptor antibodies, e.g. rhuMAb and RHUFab, VEGF aptamer e.g.Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgG1 antibody,Angiozyme (RPI 4610) and Bevacizumab (Avastin™)

Photodynamic therapy as used herein refers to therapy which uses certainchemicals known as photosensitizing compounds to treat or preventcancers. Examples of photodynamic therapy include treatment withcompounds, such as e.g. VISUDYNE™ and porfimer sodium.

Angiostatic steroids as used herein refers to compounds which block orinhibit angiogenesis, such as, e.g., anecortave, triamcinolone.hydrocortisone, 11-α-epihydrocotisol, cortexolone,17α-hydroxyprogesterone, corticosterone, desoxycorticosterone,testosterone, estrone and dexamethasone.

Implants containing corticosteroids refers to compounds, such as e.g.fluocinolone, dexamethasone.

“Other chemotherapeutic compounds” include, but are not limited to,plant alkaloids, hormonal compounds and antagonists; biological responsemodifiers, preferably lymphokines or interferons; antisenseoligonucleotides or oligonucleotide derivatives; shRNA or siRNA; ormiscellaneous compounds or compounds with other or unknown mechanism ofaction.

The structure of the active compounds identified by code nos., genericor trade names may be taken from the actual edition of the standardcompendium “The Merck Index” or from databases, e.g. PatentsInternational (e.g. IMS World Publications).

None of the quotations of references made within the present disclosureis to be understood as an admission that the references cited are priorart that would negatively affect the patentability of the presentinvention.

The above-mentioned compounds, which can be used in combination with thecrystalline form of(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-oneas described herein, can be prepared and administered as described inthe art.

Administration

The invention also provides a pharmaceutical preparation, comprising thecrystalline form of(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-oneas described herein, and at least one pharmaceutically acceptablecarrier.

Said crystalline form as described herein can be administered alone orin combination with one or more other therapeutic compounds, possiblecombination therapy taking the form of fixed combinations or theadministration of a compound of the invention and one or more othertherapeutic (including prophylactic) compounds being staggered or givenindependently of one another, or the combined administration of fixedcombinations and one or more other therapeutic compounds. Said solidform can besides or in addition be administered especially for tumortherapy in combination with chemotherapy, radiotherapy, immunotherapy,phototherapy, surgical intervention, or a combination of these.Long-term therapy is equally possible as is adjuvant therapy in thecontext of other treatment strategies, as described above. Otherpossible treatments are therapy to maintain the patient's status aftertumor regression, or even chemopreventive therapy, for example inpatients at risk.

The dosage of the active ingredient depends upon a variety of factorsincluding type, species, age, weight, sex and medical condition of thepatient; the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound employed. A physician, clinician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentration of drug withinthe range that yields efficacy requires a regimen based on the kineticsof the drug's availability to target sites. This involves aconsideration of the distribution, equilibrium, and elimination of adrug.

The dose of the crystalline form of(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-oneas described herein to be administered to warm-blooded animals, forexample humans of approximately 70 kg body weight, is preferably fromapproximately 3 mg to approximately 15 g, more preferably fromapproximately 10 mg to approximately 3 g, yet more preferably fromapproximately 50 mg to 1.5 g per person per day, undivided in 1 dose ordivided preferably into 2 to 4, e.g. 2 or 3, single doses which may, forexample, be of the same size. Usually, children receive half of theadult dose.

Said solid form may be administered by any conventional route, inparticular parenterally, for example in the form of injectable solutionsor suspensions, enterally, e.g. orally, for example in the form oftablets or capsules, topically, e.g. in the form of lotions, gels,ointments or creams, or in a nasal or a suppository form. Topicaladministration is e.g. to the skin. A further form of topicaladministration is to the eye. Pharmaceutical compositions comprising acompound of the invention in association with at least onepharmaceutical acceptable carrier or diluent may be manufactured inconventional manner by mixing with a pharmaceutically acceptable carrieror diluent.

The invention relates also to pharmaceutical compositions comprising aneffective amount, especially an amount effective in the treatment of oneof the above-mentioned disorders, of the crystalline form of(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-oneas described herein together with one or more pharmaceuticallyacceptable carriers that are suitable for topical, enteral, for exampleoral or rectal, or parenteral administration and that may be inorganicor organic, solid or liquid.

There can be used for oral administration especially tablets or gelatincapsules that comprise the active ingredient together with diluents, forexample lactose, dextrose, mannitol, and/or glycerol, and/or lubricantsand/or polyethylene glycol. Tablets may also comprise binders, forexample magnesium aluminum silicate, starches, such as corn, wheat orrice starch, gelatin, methylcellulose, sodium carboxymethylcelluloseand/or polyvinylpyrrolidone, and, if desired, disintegrators, forexample starches, agar, alginic acid or a salt thereof, such as sodiumalginate, and/or effervescent mixtures, or adsorbents, dyes, flavoringsand sweeteners. It is also possible to use the pharmacologically activecompounds of the present invention in the form of parenterallyadministrable compositions or in the form of infusion solutions. Thepharmaceutical compositions may be sterilized and/or may compriseexcipients, for example preservatives, stabilisers, wetting compoundsand/or emulsifiers, solubilisers, salts for regulating the osmoticpressure and/or buffers. The present pharmaceutical compositions, whichmay, if desired, comprise other pharmacologically active substances areprepared in a manner known per se, for example by means of conventionalmixing, granulating, confectionning, dissolving or lyophilisingprocesses, and comprise approximately from 1% to 99%, especially fromapproximately 1% to approximately 20%, active ingredient(s).

Synthetic Route Example 106(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one

A sealable reaction flask was charged with potassium phosphate (4.44 g,20.29 mmol), evacuated and heated for 15 min at 170° C. The reactionflask was back-filled with argon at RT and Intermediate 75.6 (3.64 g,10.15 mmol), Intermediate 106.1 (5.48 g, 12.18 mmol), dioxan (75 mL) and(+/−)-trans-1,2-diaminocyclohexane (0.37 mL, 3.04 mmol) were addedsubsequently. The reaction flask was carefully evacuated under vacuum(2×) and backfilled with argon (2×) and copper (I) iodide (0.586 g, 3.04mmol) were added. The reaction mixture was stirred for 22.5 h at 95° C.The mixture was extracted between EtOAc (3×) and water (3×). The organicphases were washed with brine and dried over Na₂SO₄, filtered andevaporated to dryness. Purification of the residue by normal phasecolumn chromatography, eluting with 100% EtOAc to 20% MeOH/EtOAcfollowed by reverse phase prep-HPLC afforded the TFA salt which wasextracted between EtOAc (2×) and 1M aqueous NaHCO₃ (1×). The organicphases were washed with brine and dried over Na₂SO₄, filtered andevaporated to dryness gave the title compound as white solid (1.59 g,2.41 mmol, 23.8%): HPLC: ^(E)t_(Ret)=4.57 min; LC-MS: m/z 659.2 [M+H]⁺;1H NMR (400 MHz, DMSO-d₆) 0.88-1.01 (m, 2H) 1.05-1.14 (m, 2 H) 1.16 (d,J=5.86 Hz, 3H) 1.21 (d, J=6.25 Hz, 3H) 1.48-1.62 (m, 1H) 1.73 (dd, 4H)2.14-2.27 (m, 1H) 2.65 (t, J=5.47 Hz, 2H) 2.76 (s, 3H) 2.85 (s, 3H) 3.02(s, 2H) 3.06-3.20 (m, 4H) 3.54 (d, 1H) 3.71 (s, 3H) 3.87 (d, J=19.53 Hz,1H) 4.39-4.47 (m, 1H) 5.92 (s, 1H) 6.54 (d, 2H) 6.81 (s, 1H) 6.87 (d,2H) 7.02 (s, 1H) 7.33 (s, 4H).

Intermediate 106.1:4-(4-{[(4-Iodo-phenyl)-methyl-amino]-methyl}-trans-cyclohexyl)-1-methyl-piperazin-2-one

To a solution of Intermediate 106.2 (13.3 g, 20.9 mmol) in dioxane (52.3mL) was added 4M dioxane solution of HCl (105 mL, 418 mmol) at 0° C. Thereaction mixture was stirred at RT for 0.5 h. The solution wasconcentrated and the residue was dissolved in MeOH (157 mL),triethylamine (27.3 ml, 196 mmol) was added drop wise at 0° C. and themixture was stirred for 1 h at RT. The reaction mixture was concentratedand the residue extracted between EtOAc (2×) and 1M aqueous NaHCO₃ (1×).The organic phases were washed with brine and dried over Mg₂SO₄,filtered and evaporated to dryness. The cure material was suspended inEt₂O (50 mL), and after stirring and sonication during 30 min, it wasfiltered on paper, washed with Et₂O (50 mL) and dryness under highvacuum to give a white powder (8.11 g, 18.0 mmol, 86%). HPLC:^(E)t_(Ret)=4.035 min; LC-MS: m/z 442.1 [M+H]⁺; ¹H NMR (400 MHz,DMSO-d₆) 0.84-1.02 (m, 2H) 1.02-1.12 (m, 2H) 1.52-1.60 (m, 1H) 1.60-1.84(m, 4H) 2.16-2.27 (m, 1H) 2.65 (t, J=5.47 Hz, 2H) 2.76 (s, 3H) 2.85 (s,3H) 3.02 (s, 2H) 3.10 (d, J=7.03 Hz, 2H) 3.14-3.20 (m, 2H) 6.47 (d, 2H)7.37 (d, 2H).

Intermediate 106.2:[[2-(tert-Butoxycarbonyl-methyl-amino)-ethyl]-(4-{[(4-iodo-phenyl)-methyl-amino]-methyl}-trans-cyclohexyl)-amino]-aceticacid methyl ester

To a suspension of Intermediate 105.3 (9.45 g, 21.6 mmol),methyl-(2-oxo-ethyl)-carbamic acid tert-butyl ester (4.11 g, 23.7 mmol)and AcOH (3.7 mL, 64.7 mmol) in DCM (108 mL) was added potion wiseNaBH(OAc)₃ (13.7 g, 64.7 mmol) at 0° C. After stirring for 1 h at RT,the reaction mixture was added carefully saturated aqueous NaHCO₃ to pH8 followed by extraction with DCM (2×). The organic phases were driedover Mg₂SO₄, filtered and evaporated, which gave the crude titleintermediate (13.3 g, 20.9 mmol, 97% with 90% purity). This material wasused for the next step without further purifications. HPLC:^(E)t_(Ret)=5.32 min; LC-MS: m/z 574.3 [M+H]⁺.

Intermediate 105.3:(4-{[(4-Iodo-phenyl)-methyl-amino]-methyl}-trans-cyclohexylamino)-aceticacid methyl ester

To a solution of Intermediate 105.4 (16.9 g, 49.2 mmol) in DMF (300 mL)was successively added potassium carbonate (14.3 g, 103 mmol) and methyl2-bromoacetate (4.77 mL, 51.7 mmol) at −10° C. The suspension wasstirred for 4.5 h at −10° C. to 10° C. The reaction mixture was dilutedwith EtOAc, the organic phase was washed with water and brine, and driedover Na₂SO₄, filtered and evaporated to dryness. Purification of theresidue by normal phase column chromatography, eluting with 100% DCM to100% EtOAc, gave the title compound as brown oil (9.45 g, 21.6 mmol,43.8%). HPLC: ^(E)t_(Ret)=4.22 min; LC-MS: m/z 417.0 [M+H]⁺.

Intermediate 105.4:(Trans-4-amino-cyclohexylmethyl)-(4-iodo-phenyl)-methyl-amine

To a solution of Intermediate 77.1 (21.9 g, 49.5 mmol) in DCM (300 mL)was added drop wise TFA (114 mL, 1484 mmol) at 0° C. The reactionmixture was stirred for 30 min at RT, then concentrated in vacuo. Theresidue was diluted with EtOAc, and adjusted to pH 9 at 0° C. byaddition of 2M NaOH. The phases were separated and water layer wasextracted with EtOAc. The organic layer was washed with water and brine,dried over Na₂SO₄, and filtered. Concentration in vacuo gave the titlecompound as grey solid (16.9 g, 47.8 mmol, 97%). HPLC: ^(E)t_(Ret)=3.92min; LC-MS: m/z 345.1 [M+H]⁺.

Intermediate 77.1:(4-{[(4-Iodo-phenyl)-methyl-amino]-methyl}-trans-cyclohexyl)-carbamicacid tert-butyl ester

To a solution of Intermediate 75.7 (748 mg, 1.74 mmol) in DCM (15 ml)were successively added AcOH (0.199 ml, 3.48 mmol), formaldehyde (37% inwater, 0.259 ml, 3.48 mmol) and NaBH(OAc)₃ (737 mg, 3.48 mmol) at RT.The reaction mixture was stirred at RT for 2 h then diluted with DCM andwashed with a 2M aqueous Na₂CO₃ solution (2×). The organic phase wasdried over Na₂SO₄, filtered and evaporated to dryness. The resultingcrude material was purified by Combi-Flash Companion™ (Isco Inc.) columnchromatography (SiO₂; gradient elution, heptane/AcOEt 98:2→7:3) to yieldthe title compound (584 mg, 1.31 mmol, 76%) as a colorless solid. TLC:R_(F)=0.36 (heptane/AcOEt 3:1); HPLC: ^(A)t_(Ret)=2.76 min; LC-MS: m/z445.4 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆): 0.90-1.13 (m, 4H), 1.36 (s,9H), 1.48-1.66 (m, 3H), 1.69-1.81 (m, 2H), 2.87 (s, 3H), 3.08-3.21 (m,1H), 3.12 (d, J=7.1, 2H), 6.45-6.54 (m, 2H), 6.68 (d, J=8.1, 1H),7.34-7.43 (m, 2H).

Intermediate 75.7:{4-[(4-Iodo-phenylamino)-methyl]-trans-cyclohexyl}-carbamic acidtert-butyl ester

To a solution of 4-iodo-phenylamine (1 g, 4.57 mmol) in DCM (25 ml) weresuccessively added AcOH (0.523 ml, 9.13 mmol),(4-formyl-cyclohexyl)-carbamic acid tert-butyl ester (1.14 g, 5.02 mmol)and NaBH(OAc)₃ (1.94 g, 9.13 mmol) at RT. The reaction mixture wasstirred at RT for 1 h then diluted with Et₂O and washed successivelywith a 2M aqueous HCl solution and a 2M aqueous Na₂CO₃ solution. Theorganic phase was dried over Na₂SO₄, filtered and evaporated to dryness.The resulting crude material was purified by Combi-Flash Companion™(Isco Inc.) column chromatography (SiO₂; gradient elution, heptane/AcOEt95:5→1:1) to yield the title compound (1.56 g, 3.62 mmol, 79%) as acolorless solid. TLC: R_(F)=0.72 (heptane/AcOEt 1:1); HPLC:^(A)t_(Ret)=2.64 min; LC-MS: m/z 431.4 [M+H]⁺; ¹H NMR (400 MHz,DMSO-d₆): 0.87-1.03 (m, 2H), 1.03-1.17 (m, 2H), 1.32-1.48 (m, 1H), 1.37(s, 9H), 1.70-1.86 (m, 4H), 2.76-2.86 (m, 2H), 3.08-3.25 (m, 1H),5.77-5.89 (m, 1H), 6.34-6.46 (m, 2H), 6.59-6.71 (m, 1H), 7.24-7.35 (m,2H).

Intermediate 75.6:(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-1,4-dihydro-2H-isoquinolin-3-one

A solution of Intermediate 75.5 (3.96 g, 7.98 mmol) in 1.25M HCl in MeOH(128 ml) was stirred at RT for 30 min. The reaction mixture wasevaporated to dryness and the resulting residue was dissolved in MeOH(40 ml). Et₃N (5.56 ml, 39.9 mmol) was added at RT then the mixture wasstirred for 15 min and evaporated to dryness. The resulting crudematerial was purified by Combi-Flash Companion™ (Isco Inc.) columnchromatography (SiO₂; gradient elution, [heptane/DCM 1:1]/TBME 9:1→100%TBME) to yield the title compound (2.51 g, 7.24 mmol, 91%, ee 92%) as anoff-white solid. TLC: R_(f)=0.13 (heptane/DCM/TBME 1:1:2); HPLC:^(A)t_(Ret)=2.03 min; LC-MS: m/z 346.4 [M+H]⁺; ¹H NMR (400 MHz,DMSO-d₆): 1.16 (d, J=6.1, 3H), 1.21 (d, J=6.1, 3H), 3.36 (d, J=19.8,1H), 3.51 (d, J=19.8, 1H), 3.72 (s, 3H), 4.40 (spt, J=6.1, 1H), 5.55 (d,J=3.4, 1H), 6.79 (s, 1H), 6.84 (s, 1H), 7.26-7.33 (m, 2H), 7.35-7.42 (m,2H), 8.49 (d, J=3.9, 1H).

Intermediate 75.5:{2-[(S)-(4-Chloro-phenyl)-((S)-2-methyl-propane-2-sulfinylamino)-methyl]-4-isopropoxy-5-methoxy-phenyl}-aceticacid ethyl ester

A 250-mL flask was charged with Intermediate 75.3 (10.97 g, 28.6 mmol)and anhydrous THF (50 ml) then evacuated under vacuum and back-filledwith argon (3×). Intermediate 75.4 (15.75 g, 57.2 mmol) andbis(acetonitrile)(1,5-cyclooctadiene)rhodium(I)tetrafluoro-borate (1.09g, 2.86 mmol) were successively added at RT and the resulting orangesuspension was heated at 60° C. and stirred for 2 h. Additionalbis(acetonitrile)(1,5-cyclooctadiene)rhodium(I)tetrafluoroborate (1.09g, 2.86 mmol) was added at 60° C. and the mixture was further stirredfor 4 h. The reaction mixture was cooled to RT, diluted with AcOEt andwashed with water. The organic phase was dried over Na₂SO₄, filtered andevaporated to dryness. The resulting crude material was purified byCombi-Flash Companion™ (Isco Inc.) column chromatography (SiO₂; gradientelution, heptane/AcOEt 95:5→3:7) to yield the title compound (3.96 g,7.98 mmol, 28%) as a brownish resin. TLC: R_(f)=0.29 (heptane/AcOEt1:1); HPLC: ^(A)t_(Ret)=2.70 min; LC-MS: m/z 496.3 [M+H]⁺; ¹H NMR (400MHz, DMSO-d₆): 1.10-1.19 (m, 15H), 1.23 (d, J=5.9, 3H), 3.57 (d, J=16.4,1H), 3.68 (d, J=16.1, 1H), 3.73 (s, 3H), 3.93-4.05 (m, 2H), 4.37-4.45(m, 1H), 5.62 (d, J=6.1, 1H), 5.82 (d, J=6.1, 1H), 6.82 (s, 1H), 6.94(s, 1H), 7.25-7.30 (m, 2H), 7.36-7.41 (m, 2H).

Intermediate 75.4: (4-Chloro-phenyl)-trimethyl-stannane

To a 1M solution of trimethyltin chloride in THF (92 ml, 92 mmol) wasslowly added a 1M solution of 4-chlorophenylmagnesium bromide in Et₂O(92 ml, 92 mmol) over a 40 min period at −10° C. so that the temperaturenever exceed 0° C. After the addition, the cooling bath was removed andthe resulting suspension was stirred at RT for 1 h. A saturated aqueoussolution of NH₄Cl (14 ml) was added followed by water until completedissolution of the precipitate. The mixture was transferred into aseparating funnel and extracted with Et₂O (3×). The combined organicfractions were dried over Na₂SO₄, filtered and evaporated to dryness.The resulting crude material was purified by Combi-Flash Companion™(Isco Inc.) column chromatography (SiO₂; isocratic elution withcyclohexane) to yield the title compound (24.47 g, 89 mmol, 97%) as acolorless oil. TLC: R_(F)=0.76 (cyclohexane/AcOEt 95:5); HPLC:^(A)t_(Ret)=3.25 min; ¹H NMR (400 MHz, CDCl₃): 0.31 (s, 9H) 7.32-7.36(m, 2H) 7.41-7.45 (m, 2H).

Intermediate 75.3:(4-Isopropoxy-5-methoxy-2-{[(E)-(S)-2-methyl-propane-2-sulfinylimino]-methyl}-phenyl)-aceticacid ethyl ester

To a solution of Intermediate 75.2 (9.14 g, 32.6 mmol) and(S)-(−)-2-methyl-2-propanesulfinamide (5.93 g, 48.9 mmol) in DCM (200ml) was added Ti(OEt)₄ (27.3 ml, 130 mmol) at 0° C. (ice bath). Thereaction mixture was heated at reflux, stirred for 5 h then cooled to RTand quenched by the careful addition of water (14.7 ml). The resultingwhite precipitate was filtered through a Celite pad, the filter cake waswashed with DCM and the filtrate then evaporated to dryness. Theresulting crude material was purified by Combi-Flash Companion™ (IscoInc.) column chromatography (SiO₂; gradient elution, heptane/AcOEt95:5→1:1) to yield the title compound (11.07 g, 28.9 mmol, 89%) as ayellow oil. TLC: R_(F)=0.40 (heptane/AcOEt 1:1); HPLC: ^(A)t_(Ret)=2.35min; LC-MS: m/z 384.5 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆): 1.17 (t, J=7.1,3H) 1.15 (s, 9H) 1.27 (d, J=6.1, 6H) 3.83 (s, 3H) 3.94-4.07 (m, 4H)4.58-4.66 (m, 1H) 7.04 (s, 1H) 7.50 (s, 1H) 8.49 (s, 1H).

Intermediate 75.2: (2-Formyl-4-isopropoxy-5-methoxy-phenyl)-acetic acidethyl ester

To a solution of Intermediate 75.1 (11.94 g, 47.3 mmol) anddichloro-methoxy-methane (8.56 ml, 95 mmol) in DCM (350 ml) was slowlyadded SnCl₄ (1M solution in DCM, 95 ml, 95 mmol) over a 45 min period at0° C. (ice bath). After the addition, the reaction mixture was furtherstirred at 0° C. for 45 min then poured into water and extracted withDCM (2×). The organic phase was washed with a 2M aqueous Na₂CO₃solution, then dried over Na₂SO₄, filtered and evaporated to dryness.The resulting crude material was purified by Combi-Flash Companion™(Isco Inc.) column chromatography (SiO₂; gradient elution, heptane/AcOEt95:5→1:1) to yield the title compound (11.13 g, 39.7 mmol, 84%) as ayellow oil which crystallized on standing into an off-white solid. TLC:R_(F)=0.50 (heptane/AcOEt 1:1); HPLC: ^(A)t_(Ret)=1.93 min; LC-MS: m/z281.4 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆): 1.18 (t, J=7.1, 3H) 1.28 (d,J=6.1, 6H) 3.84 (s, 3H) 4.01 (s, 2H) 4.07 (q, J=7.1, 2H) 4.56-4.68 (m,1H) 7.03 (s, 1H) 7.45 (s, 1H) 9.93 (s, 1H).

Intermediate 75.1: (4-Isopropoxy-3-methoxy-phenyl)-acetic acid ethylester

A mixture of ethyl (4-hydroxy-3-methoxy-phenyl)-acetic acid ethyl ester(11.22 g, 53.4 mmol) and K₂CO₃ (22.13 g, 160 mmol) in DMF (100 ml) washeated at 60° C. 2-Iodopropane (9.06 ml, 91 mmol) was added and themixture was vigorously stirred at 60° C. for 5 h. The reaction mixturewas cooled to RT, diluted with AcOEt and washed with water. The aqueousphase was separated and further extracted with AcOEt. The combinedorganic fractions were dried over Na₂SO₄, filtered and evaporated todryness. The resulting crude material was purified by Combi-FlashCompanion™ (Isco Inc.) column chromatography (SiO₂; gradient elution,heptane/AcOEt 98:2→3:1) to yield the title compound (11.94 g, 47.3 mmol,89%) as a colorless oil. TLC: R_(F)=0.44 (heptane/AcOEt 7:3); HPLC:^(A)t_(Ret)=2.14 min; LC-MS: m/z 253.4 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃):1.28 (t, J=7.1, 3H), 1.38 (d, J=6.1, 6H), 3.56 (s, 2H), 3.87 (s, 3H),4.17 (q, J=7.1, 2H), 4.50 (h, J=6.1, 1H), 6.77-6.89 (m, 3H).

Process to Make the Crystalline Form I of the Sulphate Salt of theCompound of Example 106: A: Slurry Method

Solvent: isopropyl alcohol(1) About 5 mg of drug substance was first dissolved in 100 μl IPA.(2) 364 μl 0.025N sulphuric acid was added to the solution very slowly,allowing slow precipitation during the stirring at 60° C.(3) The suspension was stirred at room temperature overnight.(4) The supernatant was removed by centrifugation.(5) The solid product was dried under vacuum oven at 40° C. overnightand investigated by XRPD (X-ray powder diffraction). The process wasscaled up, and scale-up samples were further characterized using XRPD.Crystalline form I was obtained.

The X-ray diffraction data were collected at room temperature using aBruker AXS GMBH D8-Discover powder X-ray diffractometer (Cu Kαradiation) fitted with an automatic sample changer, a theta-thetagoniometer, automatic beam divergence slits, a secondary monochromatorand a scintillation counter. Samples were prepared for analysis bygently pressing the compound in one glass filter. The sample was rotatedwhile being irradiated with Copper Kα1 X-rays (wavelength=1.54184Ångstroms) with the x-ray tube operated at 40 kV/40 mA. The analyseswere performed with the goniometer running in continuous mode set for a120 second count per 0.02 degree step over a two theta range of 5 degreeto 45 degree. The peaks obtained were aligned against the siliconreference standard.

Instrument Name: X-Ray Diffractometer Model: D8 Discover Manufacturer:Bruker AXS GMBH Wavelength: 1.54184 A (Cu)

Generator setting: 40.00 KV, 40.00 mA

Monochromator Detector: HI-STAR

Frame Size: 1024 pixels, 107.79 mm

Experiment Method:

2-Theta start: 5.0 degree2-Theta end: 45.0 degreePixel overlap: 20%Integration stepsize: 0.02 degreeScan time: 120 seconds

Temperature: Room Temperature

TABLE A XRPD data of Example 106 sulphate salt crystalline form I (A:slurry method) Angle d value Intensity % 2-Theta ° Angstrom % 17.1 5.20126 18.7 4.74 103 20.4 4.35 89.2 21.4 4.14 93.5 22.9 3.89 183 23.5 3.78111 24.1 3.68 132 28.3 3.15 88.9

B: Anti-Solvent Method

Solvents: isopropyl alcohol(1) About 5 mg of drug substance was first dissolved in 91 μl 0.025Nsulfuric acid IPA.(2) Anti-solvent methyl tert-butyl ether was added to precipitate thecompound during the stirring at 55-60° C.(3) The suspension was stirred at 55-60° C. overnight.(4) By centrifugation, the supernatant was removed.(5) The solid product was dried in the vacuum oven at 40° C. overnightand investigated by XRPD. The process was scaled up. Scale-up sampleswere further characterized using XRPD. Crystalline form I was obtained.

(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-onebisulphate monohydrate was formed using this method.

TABLE B XRPD data of Example 106 sulphate salt crystalline form I (B:Anti-solvent method) Angle d value Intensity % 2-Theta ° Angstrom % 13.56.56 89.1 16.6 5.35 117 16.9 5.24 226 18.8 4.73 114 19.8 4.48 167 21.34.17 117 22.7 3.92 270 23.9 3.72 172 24.9 3.57 180 error +/−0.2°.

It will be appreciated by the skilled crystallographer that the relativeintensities of the various peaks reported in the Tables and Figures mayvary due to a number of factors such as the orientation effects of thecrystals in the X-ray beam, and the purity of the material beinganalysed. The peak positions may also shift for variations in sampleweight but will remain substantially the same.

The sulphate salt formed is believed to be the bisulphate salt.

The invention also relates to the manufacture of Example 106 sulphatesalt crystalline form I, as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 discloses the X-ray powder diffraction data for Example 106sulphate salt crystalline form I, as obtained using the slurry method.

FIG. 2 discloses the X-ray powder diffraction data for Example 106sulphate salt crystalline form I, as obtained using the anti-solventmethod.

1. A crystalline form of (S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one sulphate salt.
 2. The crystalline form of (S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one sulphate salt as in claim 1, wherein the sulphate salt is the bisulphate salt.
 3. The crystalline form of (S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one bisulphate salt, of claim 1, having a powder X ray diffraction pattern using Cu Kα radiation which includes the peaks: 18.8, 21.3 and 22.7 angle 2-Theta°, with an error +/−0.2°.
 4. The crystalline form of (S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one bisulphate salt, of claim 1, having a powder X ray diffraction pattern using Cu Kα radiation which is substantially the same as as the powder X ray diffraction pattern shown in FIG. 1 herein.
 5. The crystalline form of (S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one bisulphate salt, of claim 1, having a powder X ray diffraction pattern using Cu Kα radiation which is substantially the same as as the powder X ray diffraction pattern shown in FIG. 2 herein.
 6. The crystalline form of claim 1 for use as pharmaceutical.
 7. A pharmaceutical composition comprising a therapeutically effective amount of a crystalline form as defined in claim 1 and one or more pharmaceutically acceptable carriers.
 8. A method of modulating MDM2 and/or MDM4 activity in a subject, comprising the step of administering to a subject a therapeutically effective amount of a crystalline form as defined in claim
 1. 9. A method for the treatment of a disorder or a disease mediated by the activity of MDM2 and/or MDM4 comprising the step of administering to a subject a therapeutically effective amount of a crystalline form as defined in claim
 1. 10. The method of claim 9, wherein the disorder or a disease is a proliferative disorder or disease.
 11. A crystalline form as defined in claim 1, in combination with one or more therapeutically active agents. 